RU2814729C2 - Novel isoform of trypsin and use thereof - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: disclosed is an isolated isoform of Atlantic cod trypsin ZT containing the amino acid sequence SEQ ID NO: 2 or amino acid sequence with sequence homology of 99% or more, a medical composition based thereon and a method of treating diseases caused by picornaviruses, which involves administering a therapeutically effective amount of the composition.

EFFECT: new isoform of trypsin ZT is better adapted for cleavage of peptides containing several sequential basic amino acid residues Arg and Lys, has specific antiviral activity.

11 cl, 5 dwg, 4 tbl, 6 ex

Description

Field of technology

The present invention relates to new isoforms of trypsin ZT. In particular, the invention relates to the use of ZT trypsin isoforms in medical devices, pharmaceuticals and cosmetics.

State of the art

Proteinases are enzymes that are defined by their ability to break down proteins. Proteinases can cleave substrates at different amino acid residues in the protein sequence, demonstrating differences in substrate specificity. Trypsins are proteinases and are defined in part by their preference for cleaving the C-terminus into arginine or lysine residues (Olsen, Ong et al., 2004, Mol Cell Proteomics 3: 608-614). Multiple substrate profiling identifies proteinase cleavage sites and reveals the critical importance of residues surrounding the cleavage site by their specificity (O'Donoghue, Eroy-Reveles et al., 2012, Nat Methods 9: 1095-1100).

US Pat. Nos. 4,801,451 and 4,963,991 describe a mixture of exo- and endopeptidases isolated from Antarctic krill (Euphasia superba) and the use of this mixture as a cleaning solution. 4,801,451 describes the use of such enzymes to remove foreign matter and dead tissue from wounds. WO 85/04809 describes the use of krill enzymes as a digestive aid. EP-A1-0170115 describes the use of krill enzymes to dissolve blood clots. However, all of these references disclose impure or poorly characterized materials. To obtain a pharmaceutically useful product, a purified peptidase or a mixture of purified peptidases is desirable.

WO 96/24371 describes the use of a multifunctional proteolytic enzyme derived from krill and a family of proteolytic enzymes derived from crustaceans and fish having structural similarities to a multifunctional enzyme derived from Antarctic krill. The document also relates to methods for purifying the multifunctional enzyme and to pharmaceutical, cosmetic and other uses of the multifunctional enzyme. Structural similarity to a multifunctional enzyme derived from Antarctic krill is defined herein as at least 70% homology to a multifunctional hydrolase derived from krill.

WO 2000078332 describes the use of cod-derived trypsins and chymotrypsins in pharmaceutical compositions or medicaments for topical and topical use for the treatment of internal diseases and disorders and the cosmetic use of such enzymes. Topical application to treat local, internal disorders without the enzyme penetrating open wounds or mucous membranes has been found to be effective. Serine proteinases described in WO 2000078332 are proteinases that have at least 90% amino acid homology with trypsin I, trypsin II, trypsin III, trypsin IV, obtained from Atlantic cod, and proteinases that are chymotrypsin, which has homology in at least 90% amino acid sequence with any of chymotrypsin A and chymotrypsin B isolated from Atlantic cod.

Complementary DNA (cDNA) encoding trypsin Y was isolated from an Atlantic cod cDNA library (Spillaert and Gudmundsdottir, 1999, Mar Biotechnol (NY) 1: 598-607). Cod trypsin Y has approximately 45% identity with the two Atlantic cod trypsins I and X (WO 2000078332). Native trypsin Y and recombinant forms of trypsin Y have been previously described (Palsdottir and Gudmundsdottir, 2007, Protein Expr Purif 51: 243-252, Palsdottir and Gudmundsdottir, 2008, Chemistry Food 111: 408-414).

Brief description of the invention

The present invention relates to new isoforms of trypsin, which are called ZT trypsin isoforms. Such isoforms are derived from fish such as Atlantic cod, or can be produced in recombinant form using protein expression systems. In addition, the present invention relates to compositions containing at least one isolated isoform of cod trypsin ZT according to the invention together with suitable excipients and carriers.

The inventors have identified unexpected, useful and unique characteristics of the new ZT trypsin isoforms compared to previously known trypsins. These characteristics provide an advantage in the application of ZT trypsin isoforms for various applications in medical devices, pharmaceuticals and cosmetics. The present invention provides trypsin isoforms ZT per se and for use in the treatment or prevention of diseases caused by pathogenic organisms. This is especially important for use in the treatment or prevention of upper respiratory tract disease caused by pathogenic microorganisms. To this end, trypsin ZT isoforms can, for example, be used in medical devices or as active ingredient(s) in a drug product. In addition, the present invention provides these isoforms of trypsin ZT for use in the treatment of wound infections and burn wounds, for use in removing dead or flaky skin from remaining healthy skin, for example, in a medical device, as a drug or in the form of a cosmetic. In addition, the present invention relates to methods for producing the cod ZT trypsin isoforms of the present invention.

- Based on multiple substrate profiling, ZT trypsin isoforms prefer cleavage at Arg residues compared to trypsin I. Trypsin I prefers cleavage at Lys residues compared to ZT trypsin isoforms.

- Multiple substrate profiling shows that different amino acid residues in the substrate surrounding the cleavage site have a large influence on cleavage (about four amino acid residues on the N-terminal side and about four amino acid residues on the C-terminal side). Trypsin ZT isoforms prefer certain amino acid residues surrounding the cleavage site compared to trypsin I.

- Arginine and lysine rich amino acid sequences are often found in viral, bacterial and parasitic sequences that are associated with infection.

- Numerical values based on standard score (Z-score) data (multiple substrate profiling data) indicate that trypsin ZT isoforms are much better adapted to cleave peptides containing multiple consecutive basic amino acid residues (Arg and Lys) compared to trypsin I Based on the evidence, ZT trypsin isoforms may act as antimicrobial agents against viruses and bacteria because they can be cleaved at clustered basic amino acid residues such as lysine and arginine.

Detailed description of the invention

The present invention relates to new isoforms of trypsin, which are called ZT trypsin isoforms. Such isoforms are derived from fish such as Atlantic cod, or can be produced in recombinant form using protein expression systems. The present invention relates to the use of such ZT trypsin isoforms for the treatment and prevention of diseases and for the cosmetic field. In particular, the invention relates to new isoforms of trypsin ZT from Atlantic cod, useful as medicines, medical devices and cosmetics.

Brief description of graphic materials

In fig. Figure 1 shows the isolation of ZT trypsin isoforms by anion exchange chromatography. Chromatogram from MonoQ separation of benzamidine-purified cod trypsin isoforms showing absorbance at 280 nm compared to elution volume. Cod trypsin isoforms were loaded onto a MonoQ column and proteins eluted with a salt gradient (dashed line). The arrow indicates the peak containing ZT trypsin isoforms. The column was equilibrated with buffer (20 mM Tris, 10 mM CaCl ₂ , pH 8.0) and the enzymes were eluted with a linear 0-150 mM NaCl gradient in 40 column volumes, a linear 150-620 mM NaCl in 6 column volumes, 620-850 mM NaCl in 10 column volumes at a flow rate of 1 ml/min.

In fig. Figure 2 shows SDS-PAGE of cod ZT trypsin isoforms. SDS-PAGE analysis of the trypsin ZT-labeled anion exchange chromatography peak from Fig. 1. Trypsin 1, trypsin 5 and trypsin 7 purify and isolate cod trypsin X isoforms. Cod trypsin X is closely related to cod trypsin I and differs by about eight amino acid residues. Proteins were separated by SDS-PAGE and stained with silver gel. The bars and numbers on the left show the migration and molecular mass in kDa of standard proteins (Dalton Mark VII-L) separated on the gel.

In fig. Figure 3 shows Western blot analysis of cod trypsin X (trypsin 1, trypsin 5 and trypsin 7) and trypsin isoform ZT. Cod trypsin X and trypsin ZT isoforms were subjected to SDS-PAGE. After transfer, samples were immunoblotted with trypsin I/X (top panels) and trypsin isoform ZT (bottom panels) reactive antibodies. This demonstrates that trypsin ZT isoforms were separated from trypsin I/X using anion exchange. As can be seen in FIG. 3, a polyclonal antibody that reacts with ZT trypsin isoforms does not cross-react with trypsin I/X, and a peptide antibody raised against trypsin I/X does not cross-react with ZT trypsin isoforms.

In fig. Figure 4 shows a Venn diagram illustrating the common and unique cleavages generated by trypsin ZT and trypsin I at three different time points in a multiple substrate profiling assay. Number of common (black) and unique cleavage sites of trypsin ZT (white left) and trypsin I (white right) after incubation for 5 min. (A), 15 min. (B) and 60 min (C). The figure shows that at all time points in the library there are common cleavage sites and unique cleavage sites for trypsin ZT and trypsin I in 124 defined peptides (each containing 14 amino acid residues). The data demonstrate that the substrate specificity of trypsin ZT is different from that of trypsin I.

In fig. Figure 5 shows the antiviral activity of trypsin ZT against enterovirus (Coxsackievirus B2). The Y axis shows the percentage of infected wells compared to control, and the X axis shows control (left bar) and ZT trypsin isoform treatment (right bar).

The authors identified new trypsin isoforms called ZT trypsin isoforms. ZT trypsin includes at least the following isoforms: Atlantic cod trypsin ZT-1 isoform, Atlantic cod trypsin isoform ZT-2, Atlantic cod trypsin isoform ZT-3 and Atlantic cod trypsin isoform ZT-4. All four isoforms of Atlantic cod ZT trypsin have similar molecular weights of approximately 25 kDa. The Atlantic cod ZT trypsin isoforms of the present invention are represented by the following amino acid sequences:

SEQ ID NO: 1

Where

X ₁ selected from I and V;

X ² is selected from Q and H;

X ₃ selected from D and E;

X ₄ selected from R and N;

X ₅ represents L;

X ₆ selected from T and P;

X ₇ selected from D and A;

X ₈ selected from E and Q;

X ₉ selected from A and S;

X ₁₀ selected from V and M;

X ₁₁ selected from A and V;

X ₁₂ is selected from Y and F;

X ₁₃ selected from A and V;

X ₁₄ is selected from Q and R;

X ₁₅ selected from L and E;

X ₁₆ selected from Y and S; And

X ₁₇ is selected from Y and F.

SEQ ID NO: 2 Atlantic cod trypsin isoform ZT-1

IVGGHECEPNSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPGMISPRMMCVGFMDGGRDVCNGDS GSPLVCEGVLTGLVSWGRGC AEPNSPG VYVKVYEFLSWIQTTLDANP

SEQ ID NO:3 Atlantic cod trypsin isoform ZT-2

IVGGHECEPNSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPGMISPRMVCAGYMDGGRDACNGDS GSPLVCEGVLTGLVSWGQGC ALPNYPG VYVKVYEYLSWIQTTLDANP

SEQ ID NO:4 Atlantic cod trypsin isoform ZT-3

IIGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPGMISPRMMCVGFMDGGRDVCNGDS GSPLVCEGVLTGLVSWGRGCAEPNS PGVYV KVYEFLSWIQTTLDANP

SEQ ID NO:5 Atlantic cod trypsin isoform ZT-4

IIGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPGMISPRMVCAGYMDGGRDACNGDSGSPLVCEGVLTGLVSWGQGC ALPNYPG VYVKVYEYLSWIQTTLDANP

The ZT trypsin isoforms of the present invention have less than 50% amino acid identity with the trypsins described in WO 2000078332 and less than 45% homology with the chymotrypsins described in WO 2000078332.

The above ZT trypsin isoforms are active variants of these trypsins, that is, variants that were activated when the N-terminus of the trypsins was cleaved off. These trypsins are proteins expressed in the pyloric caeca (pancreatic tissue in fish) with a number of N-terminal amino acids that are important for secretion from cells and for maintaining the enzyme in an inactive state. Full-length trypsin ZT isoforms are also described herein as

SEQ ID NO: 19 Uncleaved isoform of Atlantic cod trypsin ZT-1

MIGLALLMLLGAAAAAVPRDVGKIVGGHECEPNSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPGMISPRMMCVGFMDGGRDVCNGDSGSPLVCEG VLTGLVSWGRGCAEPNSPGVYVKVYEFLSWIQTTLDANP

SEQ ID NO: 20 Uncleaved isoform of Atlantic cod trypsin ZT-2

MIGLALLMLLGAAAAAVPRDVGKIVGGHECEPNSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPGMISPRMVCAGYMDGGRDACNGDSGSPLVCEGV LTGLVSWGQGCALPNYPGVYVKVYEYLSWIQTTLDANP

SEQ ID NO: 21 Uncleaved isoform of Atlantic cod trypsin ZT-3

MIGLALLMLLGAAAAVPREDGRIIGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPGMISPRMMCVGFMDGGRDVCNGDSGSPLVCEGV LTGLVSWGRGCAEPNSPGVYVKVYEFLSWIQTTLDANP

SEQ ID NO: 22 Uncleaved isoform of Atlantic cod trypsin ZT-4

MIGLALLMLLGAAAAVPREDGRIIGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPGMISPRMVCAGYMDGGRDACNGDSGSPLVCEGVLT GLVSWGQGCALPNYPGVYVKVYEYLSWIQTTLDANP

Multiple substrate profiling is used to analyze the substrate specificity of enzymes (proteinases) (O'Donoghue, Eroy-Reveles et al., 2012, Nat Methods 9: 1095-1100). Proteinases can cleave substrates at different amino acid residues in the peptide sequence, demonstrating differences in substrate specificity. Trypsins are identified by their preference for cleaving the C-terminus into arginine or lysine residues. Surprisingly, multiple substrate profiling of trypsin ZT isoforms revealed that the amino acids surrounding arginine or lysine in the substrate have a different effect on its degradation compared to trypsin I. In brief, trypsin ZT isoforms or trypsin I were incubated (5, 15 and 60 min. ) with a peptide library (124 defined peptides, 14-mer sequences, total 1612 peptide bonds) with extensive physicochemical diversity (O'Donoghue, Eroy-Reveles et al., 2012, Nat Methods 9: 1095-1100). After incubation, cleavage sites were identified by mass spectrometric analysis. The results are presented in the following examples and show that there are many cleavage sites unique to ZT trypsin compared to trypsin I. Additionally, cleavage sites were identified that showed preferential cleavage by ZT trypsin isoforms compared to trypsin I. Additionally, new trypsin ZT isoforms have different substrate specificities compared to trypsin I described in WO 2000078332. These results were unexpected. Based on the results of the multiple substrate profiling analysis, a table was created containing numerical values based on the standard score value (Z-score) at positions P4-P4' in the substrate, where the substrate specific trypsin ZT was compared with trypsin I. Positive values (Z-score ) reflect the preference of an amino acid residue at a certain position P in favor of trypsin ZT isoforms compared to trypsin I.

Trypsin ZT isoforms have specific antimicrobial properties. Based on the values obtained and presented in the examples, it can be seen that trypsin ZT isoforms are much better adapted in cleavable peptides containing several consecutive positively charged residues (arginine or lysine) compared to trypsin I. These types of sequences (lysine- and arginine-rich amino acids sequences) are often found in viral proteins (Suzuki, Orba et al., 2010, PLoS Pathog 6: e1000801, Jiang, Cun et al., 2012, J Virol 86: 7256-7267, Gallaher and Garry, 2015, Viruses 7: 285 -305).

Viroporins are a group of proteins that interact with plasma membranes to alter permeability and may facilitate the release of viral particles. Clustered lysine and/or arginine residues are important for viroporin activity. Based on multiple substrate profiling analysis, ZT trypsin isoforms may act as antiviral agents against RNA viruses because they can cleave these clustered basic amino acid residues. Viroporins have been described as present in a number of virus families, including Flaviviridae, Picornaviridae, Retroviridae, Coronaviridae, Reoviridae and Paramyxoviridae (Royle, Dobson et al., 2015, Viruses 7: 5375-5387). Research has shown that the basic lysine or arginine residues in viroporins are a critical requirement for their function. Most viroporins have been identified in RNA viruses. Viruses containing lysine- and arginine-rich viroporins that would be targeted by ZT trypsin isoforms include human immunodeficiency virus (HIV), rotavirus, Ebola, Reston virus (RESTV), Lloviu virus (LLOV), Sudan virus (SUDV), Bundibugyo (BDBV), Tai Forest virus (TAFV) (Suzuki, Orba et al., 2010, PLoS Pathog 6: e1000801, Gallaher and Garry, 2015, Viruses 7: 285-305).

Viroporins are found in a wide range of DNA and RNA viruses, such as Paramecium bursaria chlorella virus 1, simian virus 40 (SV40), human polyoma JC (JCV), severe acute respiratory syndrome coronavirus (SARS-CoV) (Kang, Moroni et al., 2004, Proc Natl Acad Sci USA 101: 5318-5324, Liao, Tam et al., 2006, Adv Exp Med Biol 581: 199-202, Daniels, Sadowicz et al., 2007, PLoS Pathog 3: e98) .

Recently, highly basic viral proteins called agnoproteins have been identified from DNA viruses such as the families Polyomaviridae and Papillomaviridae (Royle, Dobson et al., 2015, Viruses 7: 5375-5387). These proteins display a number of viroporin characteristics. Conserved sequences have been identified in agnoproteins, which contain a number of clustered basic amino acid residues (Royle, Dobson et al., 2015, Viruses 7: 5375-5387). Based on multiple substrate profiling analysis, ZT trypsin isoforms may act as antiviral agents against DNA viruses because they can cleave these clustered basic amino acid residues (Royle, Dobson et al., 2015, Viruses 7: 5375-5387).

Many viruses and bacteria have developed mechanisms to enter cells using cell entry peptides or proteins (Milletti, 2012, Drug Discov Today 17: 850-860). Viruses include pestiviruses of the family Flaviviridae, herpes virus (HSV-1), human immunodeficiency virus (HIV-1, hepatitis B and human respiratory syncytial virus (RSV) (Elliott and O'Hare, 1997, Cell 88: 223-233, Oess and Hildt, 2000, Gene Ther 7: 750-758, Langedijk, 2002, J Biol Chem 277: 5308-5314, Langedijk, Olijhoek et al., 2005, International Congress Series 1277: 95-107, Lu, Tager et al. , 2006, Anal Biochem 353: 7-14, Godet, Guergnon et al., 2010, PLoS One 5: e13760) Additionally, these types of proteins have been found in bacteria such as Mycobacterium tuberculosis (Lu, Tager et al., 2006, Anal Biochem 353: 7-14) Cell penetration peptides (CPPs) have in common that they are rich in basic amino acid residues, especially arginines (Milletti, 2012, Drug Discov Today 17: 850-860).Porins are proteins outer membrane, which play a crucial role in bacterial pathogenicity and defense, and are for this reason useful targets for therapy (Galdiero, Falanga et al., 2012, Curr Protein Pept Sci 13: 843-854). These proteins often contain consecutive arginine amino acid residues. Based on multiple substrate profiling analysis, trypsin ZT isoforms may act as antimicrobial agents against viruses and bacteria because it is able to cleave clustered basic amino acid residues.

Parasites depend on virulence determinants such as proteins containing arginine-rich domains, such as Toxoplasma gondii (Fentress, Steinfeldt et al., 2012, Cell Microbiol 14: 1921-1933). Multiple substrate profiling analysis indicated that ZT trypsin isoforms would be beneficial against parasites by acting against such proteins.

Basic Local Alignment Search Tool (BLAST) was used to search for some of the unique and preferred amino acid sequences digested by ZT trypsin isoforms. The search results showed that these types of sequences are found in many pathogens, including viruses (eg, from the Paramyxoviridae and Coronaviridae virus families), bacteria (eg, the genus Haemophilus), and parasites (eg, the genus Plasmodium). The family Paramyxoviridae includes viruses such as respiratory syncytial virus (RSV) and the human paravirus viruses and the viruses that cause mumps and measles. The family Coronaviridae includes the viruses that cause severe acute respiratory syndrome (SARS). https://en.wikipedia.org/wiki/Severe_Acute_Respiratory_Syndrome The genus Haemophilus bacteria includes Haemophilus parainfluenzae, which is associated with endocarditis, meningitis, and bacteremia. The genus Plasmodium includes Plasmodium falciparum, which causes malaria.

The phrase "medical device" means, but is not limited to, any instrument, apparatus, device, software, material or other article for the purpose of diagnosis, prevention, monitoring, treatment or alleviation of disease, such as diagnosis, monitoring, treatment, alleviation or compensation; for injury or defect, such as exploration, replacement or modification of anatomy or physiological process; concept control; including devices that do not achieve their primary intended effect in or on the human body by pharmacological, immunological or metabolic means, but can be exploited to their function by such means.

The present invention relates to new isoforms of trypsin ZT. The invention describes the unpredictable and unique protein cleavage properties of ZT trypsin isoforms that make it valuable in medical applications. In particular, the invention relates to new isoforms of trypsin ZT from Atlantic cod, useful as medicines, medical devices and cosmetics.

In one aspect of the invention, an isolated cod ZT trypsin isoform is provided comprising an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence with 80% or greater sequence homology. Typically, such sequence homology is 85%, 90%, 95%, or 99% homology to the amino acid sequence of SEQ ID NO: 1.

In one embodiment of this aspect, an isolated cod ZT trypsin isoform is provided selected from ZT-1 trypsin containing the amino acid sequence of SEQ ID NO: 2; trypsin ZT-2 containing the amino acid sequence in accordance with SEQ ID NO: 3; trypsin ZT-3 containing the amino acid sequence according to SEQ ID NO: 4; and trypsin ZT-4 containing an amino acid sequence in accordance with SEQ ID NO: 5 or an amino acid sequence with 80% or more sequence homology. Also included are amino acid sequences with 80% sequence homology or more to the amino acid sequences shown in SEQ ID NO: 2-5. Typically, such sequence homology is 85, 90, 95 or 99% homology to the amino acid sequence of SEQ ID NO: 2-5.

In one aspect of the invention, a composition is provided comprising at least one cod ZT trypsin isoform according to the invention together with suitable excipients and carriers. Said cod ZT trypsin isoform may be selected from cod ZT-1 trypsin containing the amino acid sequence of SEQ ID NO: 2; cod trypsin ZT-2 containing the amino acid sequence according to SEQ ID NO: 3; cod trypsin ZT-3 containing the amino acid sequence according to SEQ ID NO: 4; and cod trypsin ZT-4 containing the amino acid sequence of SEQ ID NO: 5. Also included are amino acid sequences with 80% sequence homology or more to the amino acid sequences shown in SEQ ID NO: 2-5. Typically, such sequence homology is 85, 90, 95 or 99% homology to the amino acid sequence of SEQ ID NO: 2-5.

Typically, said composition contains cod ZT trypsin isoforms present in a mixture with other cod trypsins. Preferably, the ZT cod trypsin isoforms contain at least 5% (w/w) of the total cod trypsin content of said composition, such as at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 % or more (wt./wt.) of the total content of cod trypsins in the specified composition.

The composition is usually applied topically, in the form of a lotion, hydrogel, oral aerosol or nasal spray. Said composition may further contain a polyvalent alcohol such as glycerin.

In one embodiment of this aspect, said composition is provided for use in therapy.

In one embodiment of this aspect, the composition is provided for the treatment or prevention of a disease caused by a pathogenic organism selected from the group consisting of a virus, a bacterium, a fungus, a parasite and a protozoan.

In one embodiment of this aspect, the composition is provided for use in the treatment or prevention of upper respiratory tract disease caused by pathogenic organisms such as viruses, bacteria and fungi.

In one embodiment of this aspect, the composition is provided for the treatment or prevention of pain, acute inflammation, chronic inflammation, arthritis, joint inflammation, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus, phlebitis, tendinitis, rashes, psoriasis, acne, eczema, seborrheic eczema of the face, eczema of the hands, face or neck, foreskin infections, athlete's foot, fistula infections, infected malignant ulcers, umbilical infections in newborns, wrinkles, scars, keloids, boils, warts and allergic itching, hemorrhoids, fungal infection and immunological disorder, including autoimmune disease.

In one embodiment of this aspect, the composition is provided for use in the treatment of wound infections and burn wounds.

In one embodiment of this aspect, the composition is provided for use in removing dead or flaky skin from remaining healthy skin.

In one embodiment of this aspect, said composition is provided for cosmetic use. Said composition may further contain an additional cosmetically active compound.

In one aspect of the invention, there is provided a method of treating a disease, comprising administering a therapeutically effective amount of a composition in accordance with the present invention to a patient in need thereof.

In one embodiment of this aspect, said disease is caused by a pathogenic organism selected from the group consisting of a virus, a bacterium, a fungus, a parasite, and a protozoan

In one embodiment of this aspect, said disease is an upper respiratory tract disease caused by pathogenic organisms such as viruses and bacteria.

In one embodiment of this aspect, said disease is selected from pain, acute inflammation, chronic inflammation, arthritis, joint inflammation, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus, phlebitis, tendinitis, rash, psoriasis , acne, eczema, seborrheic eczema of the face, eczema of the hands, face or neck, foreskin infections, athlete's foot, fistula infections, infected malignant ulcers, umbilical infections in newborns, wrinkles, scars, keloids, boils, warts and allergic itching, hemorrhoids, fungal infection and immunological disorder, including autoimmune disease.

In one embodiment of this aspect, said disease is a wound infection or burn wound.

In one aspect of the invention, an isolated cod ZT trypsin isoform is provided selected from full-length ZT-1 trypsin containing the amino acid sequence of SEQ ID NO: 19; full-length trypsin ZT-2 containing the amino acid sequence according to SEQ ID NO: 20; full-length trypsin ZT-3 containing the amino acid sequence in accordance with SEQ ID NO: 21; and full-length trypsin ZT-4 containing the amino acid sequence according to SEQ ID NO: 22.

In one aspect of the invention, there is provided a method for producing cod ZT trypsin isoforms comprising an amino acid sequence according to any one of SEQ ID NO: 2-5, comprising:

- extraction of trypsin ZT from cod entrails,

- using the extract in at least one or more chromatography steps, including an affinity chromatography step using the affinity ligand p-aminobenzamidine and

- desorption and elution of ZT trypsin isoforms bound to the affinity ligand p-aminobenzamidine.

In one embodiment of this aspect, said method further includes at least one step using an anion exchange resin after said affinity chromatography step (ii).

In one embodiment of this aspect, said cod ZT trypsin isoforms are selected from ZT-1 trypsin containing the amino acid sequence of SEQ ID NO: 2; trypsin ZT-2 containing the amino acid sequence in accordance with SEQ ID NO: 3; trypsin ZT-3 containing the amino acid sequence according to SEQ ID NO: 4; and trypsin ZT-4 containing the amino acid sequence in accordance with SEQ ID NO: 5 or a mixture thereof.

Cod intestines are commonly used for the purification and isolation of ZT trypsin isoforms. We have identified new methods, described below, suitable for the commercial production of ZT trypsin isoforms. Extraction is carried out at pH 6-8 at temperatures below 10°C. Cod entrails are mixed with water in a ratio (w/w) from 1:6 to 1:20. The extract is separated from residual by-products and further clarified by sedimentation and filtration. This is followed by microfiltration and ultrafiltration, resulting in a solution containing ZT trypsin isoforms, which is used for chromatographic separation, which may involve several steps.

To purify the ZT trypsin isoforms, affinity chromatography is performed, preferably aminobenzamidine affinity chromatography. ZT trypsin isoforms can then be further purified by anion exchange chromatography.

One method of using ZT trypsin isoforms or mixtures of purified cod trypsins containing ZT trypsin isoforms is to formulate a hydrogel or lotion and water containing from 0 to 90% (v/v) of a polyvalent alcohol (polyol), such as glycerol. A suitable concentration of ZT trypsin isoforms is at least 1 to 100 the ratio of protein concentration to other forms of trypsin, preferably 5% or higher (w/w) of the total cod trypsin content. Trypsin activity ranges from 0.1 to 10,000 activity units for CBZ-Gly-Pro-Arg-pNA (carbobenzoxy Gly-Pro-Arg-pair nitroanilide) per 100 milliliters of the final hydrogel/lotion preparation.

The invention further provides (a) methods, subject to specific conditions, using effective amounts of the purified ZT trypsin isoforms described above, (b) compositions or substances for use in such methods, (c) pharmaceutical or medical compositions containing effective amounts of the trypsin isoform ZT for use in such methods, and (d) use of an enzyme or enzyme composition for the manufacture of a medicinal product (pharmaceutical and medical device) for use in such methods.

These methods include, but are not limited to:

treatment or prevention of pathogenic disease caused by viruses, bacteria and fungi, which, for example, occur in the upper respiratory tract, lower respiratory tract and lungs;

treatment or prophylactic prevention of dermatological conditions such as acne, rash, psoriasis or eczema, including seborrheic eczema of the face or eczema of the hands, face, scalp or neck, hemorrhoids and the like, wherein a preferred amount of ZT trypsin isoforms and other trypsins administered treat the dermatological condition or prevent an effective amount;

treatment of pulmonary diseases where ZT trypsin isoforms with or without other trypsins are used, for example, in a high-performance metered dose inhaler at a treatment-effective dose;

treatment or prophylactic prevention of wound infection and wound cracking (by administering an antibacterial infection to the wound, preventing an effective amount of ZT trypsin isoforms with or without other trypsins, or by promoting wound healing by administering a microbe-inhibiting effective amount of ZT trypsin isoforms with or without other trypsins, in the treatment the wound can be substantially cleared of necrotic tissue;

treatment of burns, where a preferred amount of ZT isoform trypsin, with or without other trypsins, is administered in sufficient quantities to promote healing;

treatment of sunburn, where a preferred amount of ZT isoform trypsin with or without other trypsins is administered in sufficient quantities to promote healing;

treatment of radiation burns, where a preferred amount of ZT isoform trypsin with or without other trypsins is administered in sufficient quantities to promote healing;

removing dead or flaky skin from remaining healthy skin to improve the appearance of the skin, wherein a preferred amount of ZT trypsin isoforms with or without other trypsins is administered in an amount effective to remove dead skin;

treatment of cracked heels, where a preferred amount of ZT trypsin isoforms with or without other trypsins is administered in an amount sufficient for effective treatment;

for the treatment of irritated skin such as dry patches, itching, redness and blemishes, wherein a preferred amount of ZT trypsin isoforms with or without other trypsins is administered in an amount sufficient for effective treatment;

treatment or prophylactic prevention of cystic fibrosis, cancer, for example by administering an effective amount to treat a tumor or tumor metastasis, an interfering or inhibitory amount of trypsin ZT isoforms, atherosclerosis, asthma, septic shock, toxic shock syndrome, tissue adhesions such as tendon-sheath, post-operative abdominal or joint adhesions, reperfusion injury, malaria, immune disorder such as autoimmune disease, apoptosis, colitis and enteritis such as Crohn's disease, where a preferred amount of ZT trypsin isoforms and related peptidases are effective;

treatment or prophylactic prevention of a microbial infection, for example, a viral infection such as rhinovirus (RV), respiratory syncytial virus (RSV), influenza, herpes infection (eg, HSV-1, HSV-2, herpes zoster or genital herpes), HIV , hepatitis, coronavirus, cytomegalovirus or papilloma virus; infections that cause gastrointestinal problems such as ulcers or diarrhea; a fungal infection, such as a systemic, cutaneous, oral, vaginal or esophageal fungal infection, including, for example, a yeast infection, including a fungal nail infection and Candida infections; microbial infection of the eye, preferably treated with ocular injections; bacterial infection, including Helicobacter pylori infection, Staphylococcus spp., methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumonia, Haemophilus influenza, Streptococcus mitis, Streptococcus spp., Klebsiella spp., Pseudomonas spp., Neisseria gonorrheae, Haemophilus spp., Chlamydia spp., syphilis and E. coli infections and bacterial infections causing chancroid; opportunistic microbial infections in immunocompromised patients, wherein the preferred amount of cod trypsin administered is a drug that helps treat the microbial infection or prevents an effective amount or has an inhibitory effect against cell-cell or cell-virus adhesion;

treatment or prophylactic prevention of symptoms selected from the group consisting of pain, inflammation, acute or chronic inflammation, arthritis, joint inflammation, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus and phlebitis, where preferred the amount cures or prevents the effective amount;

removing plaque, wherein a preferred amount of ZT trypsin isoforms with or without other trypsins is administered in an amount effective to effectively remove plaque; And

lysis of blood clots, wherein the preferred amount of trypsin isoform ZT is the amount effective for clot lysis.

The method involves administering a composition containing ZT trypsin isoforms, as described above.

The invention relates to topical cosmetic and medical compositions containing the ZT trypsin isoforms described above; and gel, cream or suppository compositions.

The invention also relates to a method of inhibiting or prophylactically preventing the transmission of a pathogenic microorganism by administering ZT trypsin isoforms with or without other trypsins. Preferably, trypsin ZT with or without other trypsins is applied to the body part that contains the primary entry point for the microorganism in question. In one embodiment, the spray, ointment, or lotion is applied to an orifice of the body involved in sexual activity, for example, to prevent the transmission of HIV or hepatitis. In another embodiment of the invention, trypsin ZT isoforms or related peptidases are administered to the upper respiratory tract, for example via an aerosol, to inhibit or prevent transmission of a common influenza virus, such as rhinovirus or coronavirus.

A method of in vitro treatment of tissue, body fluid, or cell composition to remove cell adhesion components reduces immune rejection of tissue, body fluid, or cell composition that is transplanted from one individual to another. In another aspect, such treatments remove or inactivate cell adhesion components found in the treated tissue, body fluid, or composition of cells involved in microbial infection.

When treating or prophylactically preventing septic shock or toxic shock syndrome by administering ZT isoforms of trypsin or related peptidases, appropriate routes of administration include systemic administration. For vaginal infections associated with shock, vaginal rinses, creams, gels, or suppositories may be used as the method of administration. For the treatment or prophylactic prevention of pain, inflammation, acute or chronic inflammation, arthritis, joint inflammation, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus, phlebitis when administered isoforms of trypsin ZT or related peptidases, appropriate routes of administration will include, without limitation, creams, gels or suppositories, in particular, but not limited to, hydrogels containing glycerol or other polyols.

For the treatment or prevention of rashes, psoriasis, acne, eczema, including seborrheic eczema of the face or eczema of the hands, face, scalp or neck, foreskin infections, athlete's foot, fistula infections, topical ulcer infections, umbilical cord infections in newborns , wrinkles, scars and keloids, boils, warts and allergic itching, hemorrhoids, etc., wounds, wound infections, burn wounds, removing dead or flaky skin from the rest of the skin to improve the skin, fungal infection such as systemic, cutaneous , oral, vaginal or esophageal fungal infection, including, for example, yeast infection, including fungal nail infection and Candida infections, and immune disorders, including autoimmune diseases, by administration of trypsin ZT isoforms with or without other trypsins, suitable routes of administration including creams, gels or suppositories, in particular, but not limited to, hydrogels containing glycerol or other polyols.

The above objectives are achieved by using a composition containing an effective amount of ZT trypsin isoforms with or without other trypsins, which is capable of relieving pain, inflammation, arthritis, swelling, hydrops, psoriasis eczema, dermatitis, rash and/or other symptoms of the disease mentioned in the introductory part. ZT trypsin isoforms can be obtained in high yield and in a relatively routine manner from cod intestines. It has been found that ZT trypsin isoforms can be purified on a commercial scale in a relatively simple manner with reasonable yields. For the purposes of the invention, trypsin ZT isoforms can be used without or together with other Atlantic serine proteases.

The ZT trypsin isoforms presented herein can be desorbed from the affinity matrix by applying conditions that destabilize the interaction between the enzyme and the affinity ligand. Such conditions include high salt content with low pH, preferably 30% or more glycerol. The column eluent is passed through a neutralization buffer to stabilize the cod trypsin after the acid elution step. ZT trypsin isoforms can then be further purified using anion exchange and eluted with increasing salt concentration. These methods can isolate ZT trypsin isoforms with a purity of over about 90%. The cod trypsin fraction obtained in this way contains 2-3 main isoforms, which are detected by SDS electrophoresis, which gives two bands, and MALDI-TOF mass spectrometric analysis. The molecular weight of ZT trypsin isoforms is approximately 25 kDa, whereas the calculated isoelectric point is 4.35–4.49 depending on the isoform.

ZT trypsin isoforms with or without other trypsins in specific preparations can be used as pharmaceuticals, in medical devices (such as chemical formulations) or in cosmetics depending on the intended use. The ZT trypsin isoforms (with or without other trypsins) according to the invention are administered topically, orally, rectally, vaginally, by instillation (for example, into the urinary tract or into fistulas), through the pulmonary route, for example, using an aerosol (for example, using a dosing pressurized inhaler (pMDI)), by applying drops to the eye, or by a systemic route, such as parenteral, including, for example, intramuscular, subcutaneous, intraperitoneal, intra-arterial or intravenous routes. The ZT trypsin isoforms are dissolved or combined with a pharmaceutically acceptable carrier or excipient in accordance with standard pharmaceutical practice. For the oral route of administration, ZT trypsin isoforms are used in the form of tablets, capsules, lozenges, chewing gum, lozenges, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. For parenteral administration, sterile solutions of ZT trypsin isoforms are usually prepared and the pH of the solutions is adjusted and buffered accordingly. For intravenous use, total solute concentration must be monitored to make the drug isotonic. For ocular administration, ointments or liquid drops can be delivered by ocular delivery systems known in the art, such as applicators or eye droppers.

For pulmonary administration, diluents and/or carriers will be selected to ensure aerosol formation. For topical applications, ZT trypsin isoforms are typically administered in a hydrogel containing 0 to 85% glycerol, such as about 20 to 30% glycerol and possibly up to 85% glycerol.

For topical treatment, a suitable dose of ZT trypsin isoforms with or without other trypsins per application is from about 0.01 μg/cm ² to about 1 mg/cm ² , preferably from about 0.1 μg/cm ² to about 0.01 mg /cm ² (using, for example, about 0.01 mg/ml enzyme gel). For systemic treatment, dosages are typically selected to maintain serum levels of the ZT isoform of trypsin with or without other trypsins from about 0.1 mg/100 ml to about 100 mg/100 ml, preferably from about 0.5 mg/100 ml to about 2. 0 mg/100 ml. In an alternative measurement of preferred systemic administration amounts, preferably from about 0.1 mg/kg to about 10 mg/kg, more preferably about 1 mg/kg, will be used. For treatment of the vaginal and urinary tract, suitable solutions for lavage/instillation of ZT trypsin isoforms will typically have a concentration of from about 1 μg/ml to about 15 mg/ml, preferably from about 100 μg/ml to about 3 mg/ml. For all treatments, the enzyme composition is typically applied from about 1 to about 10 times per day, preferably from about 2 to about 5 times per day. Of course, these values will vary depending on a number of factors, including the type and severity of the disease, as well as the age, weight and health of the patient, as recognized by medical experts. It is believed that significantly higher doses can be used without significant side effects.

For wound healing, ZT trypsin isoforms with or without other trypsins are preferably applied more frequently than simply while the wound is first covered. Preferably, ZT trypsin isoforms are used at least every time the wound dressing is changed. ZT trypsin isoforms can also be administered at least every day, more preferably every day or several times a day. For dermatological situations such as eczema, psoriasis and the like, trypsin isoform ZT hydrogel (with or without other trypsins) is preferably applied every day, more preferably twice a day. For acute or chronic inflammation, arthritis, joint inflammation, bursitis, osteoarthritis, etc., ZT trypsin isoforms are preferably applied daily, more preferably twice daily.

Numerous methods are known in the art for determining the percentage homology of proteins. Percent identity was calculated using ClustalW2 (version Clustal2.1) when performing sequence alignments (EMBL-EBI website http://www.ebi.ac.uk/Tools/msa/clustalw2/ date 4th May 2015). http: //www.ebi.ac.uk/Tools/msa/clustalw2/

The invention will now be described by several non-limiting examples.

Example 1

Preparation of a protease mixture from cod

About 100 kg of frozen Atlantic cod was thawed and added to four times the volume of cold drinking water in the extraction tank, and the pH was adjusted to about pH 6 with sodium hydroxide solution. The mixture was stirred for about 10 hours at a temperature of 0 to 5°C. After a short period of wet sedimentation (about 30 minutes), the aqueous extract was drawn away from the remaining insoluble internal organs using a pump and collected in a sediment reservoir. The aqueous extract was left to stand in the cooled sedimentation tank for about 40-80 hours. The supernatant was decanted from the supernatant reservoir into a storage reservoir using a pump. The supernatant was concentrated 10-20 times by ultrafiltration and diafiltered to an acceptable level of ionic strength with a conductivity below about 2.5 mS/cm. About 10-15 liters of ultrafiltered and diafiltered protein concentrate were obtained.

Example 2

Purification of cod ZT trypsin isoforms from concentrated cod viscera extract

About 12 liters of the ultrafiltered and diafiltered concentrate obtained in Example 1 were applied to a continuous connected series of about 1 liter chromatography columns, the first of which contained a carboxymethyl (CM) fast cation exchange resin (GE healthcare) and the second a p-aminobenzamidine affinity ligand coupled to Sepharose resin (GE healthcare). CM columns and p-aminobenzamidine columns were pre-equilibrated with about 10 column volumes of 25 mM Tris buffer, pH 7.8, containing 2.5 mM calcium chloride (buffer A). The concentrate was pumped into SM columns and p-aminobenzamidine columns at a flow rate of about 100 ml/min. When application of the concentrated solution to the columns was completed, the residual material was washed off the continuous column system with about 8 liters of Buffer A. Once the wash was complete, the p-aminobenzamidine affinity column was disconnected from the CM column and washed with about 5 column volumes of 25 mM Tris high saline. -buffer with pH 7.5 containing 0.5 M NaCl and 2.5 mM calcium chloride. Cod trypsins were then desorbed from the affinity ligand and the column was eluted with an acidic solution of 25 mM acetic acid, pH 3.2, containing 10 mM calcium chloride and 30% glycerol.

The preparation containing purified ZT trypsin isoforms was homogeneous by SDS-PAGE electrophoresis. The purified preparation is sterilized by filtration through a 0.22-micron filter and stored frozen at a temperature of about -20°C. The preparation containing cod trypsin ZT isoform is sterilized by filtration through a 0.22 μm filter and stored frozen at about -20°C. A preparation containing the cod ZT isoform of trypsin was used in an anion exchange chromatography (MonoQ) column and proteins eluted with a salt gradient (Figure 1). The column was equilibrated with buffer (20 mM Tris, 10 mM CaCl ₂ , pH 8.0) and the enzymes were eluted with a linear 0-150 mM NaCl gradient in 40 column volumes, a linear 150-620 mM NaCl in 6 column volumes, 620-850 mM NaCl in 10 column volumes at a flow rate of 1 ml/min. Proteins in the ZT trypsin-labeled peak in Fig. 1 were subjected to SDS-PAGE analysis (Fig. 2). The protein band on the gel from the ZT trypsin peak was excised from the gel and analyzed using matrix-assisted laser ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Mass spectrometry (MS) analysis showed identity to trypsin ZT-1, trypsin ZT-2, trypsin ZT-3, and trypsin ZT-4 (Table 1 and Table 2). Antibodies against trypsin I and trypsin X were raised in rabbits against the peptide ((NH ₂ -) CVLSGWVRDTMA (-COOH)) (SEQ ID NO: 23) corresponding to residues 228-239 at the C-terminal end of cod trypsin I and cod trypsin X . Antibodies were affinity purified from rabbit serum using a peptide coupled to a gel bead support. Polyclonal antibodies against ZT trypsin isoforms were generated in mouse ascites fluid as described in Overkamp et al. (Overkamp, Mohammed-Ali et al., 1988, J Immunoassay 9: 51-68). A purified form of recombinant trypsin ZT-4 was injected intraperitoneally (ip) into Balb/C2 mice. Mice were sacrificed on day 34 and ascites fluid was collected. The corresponding antibodies were used in Western blot analysis. Western blot analysis was performed on proteins in the peak labeled with trypsin ZT in Fig. 1 (Fig. 3). The trypsin ZT isoform antibody reacted with peak trypsin ZT-tagged proteins, whereas the trypsin I and trypsin X antibodies did not (Fig. 3).

Table 1 presents the peptide masses obtained from MS analysis of the protein band from the SDS-PAGE gel in Fig. 2 (non-guanidated sample), see column 1 Mr (expt). Mr denotes relative molecular weight in Da. Columns 2, 3 and 4 show the calculated values (for masses corresponding to the masses in column 1) from in silico trypsin digestion of the amino acid sequences of trypsin ZT-1, trypsin ZT-2, trypsin ZT-3 and trypsin ZT-4. Column 5 shows the peptide sequence that gives the mass value for in silico digestion. Column 6 shows modifications of various peptides based on in silico digestion.

Table 1

Mr (expt) Trypsin ZT-4 Trypsin ZT-3 Trypsin ZT-2 Trypsin ZT-1 List of sequences Changes 842.4077 1045.4976 1082.5603 1144.4826 1144.5415 1144.5415 (R)IIGGQDCEPR(S) Carbamidomethyl (C) 1149.5283 1149.6150 1149.6150 1149.6150 1149.6150 (R)VFEGTEQLVK(T) 1212.4511 1216.4408 1216.4908 1216.4908 (R)MVCAGYMDGGR(D) Carbamidomethyl (C) 1260.4492 1260.4992 1260.4992 (R)MMCVGFMDGGR(D) Carbamidomethyl (C) 1262.4221 1262.4962 1262.4962 (R)MVCAGYMDGGR(D) 2 Oxidation (M)
Propionamid (C) 1276.4272 1276.4941 1276.4941 (R)MMCVGFMDGGR(D) Carbamidomethyl (C)
Oxidation (M) 1320.5165 1320.6253 1320.6253 (R)GCAEPNSPGVYVK(V) 1354.5907 1377.5624 1377.6467 1377.6467 (R)GCAEPNSPGVYVK(V) Carbamidomethyl (C) 1493.5870 1638.7873 1707.6807 1839.8099 1994.9057 2800.2263 2800.3831 2800.3831 (R)LQCLDVPIVEPVAC QASYPGMISPR(M) 2 Carbamidomethyl (C) 2814.2398 2814.3987 2814.3987 (R)LQCLDVPIVEPVAC QASYPGMISPR(M) Carbamidomethyl (C) Propionamide (C) 2830.2763 2830.3936 2830.3936 (R)LQCLDVPIVEPVAC QASYPGMISPR(M) Carboxymethyl (C) Oxidation (M) Propionamide (C) 2847.3361 2847.3725 2847.3725 (R)LQCLDVPIVETVDC EAAYPGMISPR(M) Carbamidomethyl (C) Propionamide (C)

The sequences in Table 1 are presented below:

Seq ID NO Amino acid sequence SEQ ID NO 6 IIGGQDCEPR SEQ ID NO 7 VFEGTEQLVK SEQ ID NO 8 LQCLDVPIVEPVACQASYPGMISPR SEQ ID NO 8 LQCLDVPIVEPVACQASYPGMISPR SEQ ID NO 8 LQCLDVPIVEPVACQASYPGMISPR SEQ ID NO 9 LQCLDVPIVETVDCEAAYPGMISPR SEQ ID NO 10 MVCAGYMDGGR SEQ ID NO 11 MMCVGFMDGGR SEQ ID NO 10 MVCAGYMDGGR SEQ ID NO 11 MMCVGFMDGGR SEQ ID NO 12 GCAEPNSPGVYVK SEQ ID NO 12 GCAEPNSPGVYVK

Table 2 shows the peptide masses obtained from MS analysis of the protein band from the SDS-PAGE gel in FIG. 2 (guanidated sample), see column 1 Mr (expt). Mr denotes relative molecular weight in Da. Columns 2, 3 and 4 show the calculated values (for masses corresponding to the masses in column 1) from in silico trypsin digestion of the amino acid sequences of trypsin ZT-1, trypsin ZT-2, trypsin ZT-3 and trypsin ZT-4. Column 5 shows the peptide sequence that gives the mass value for in silico digestion. Column 6 shows modifications of various peptides based on in silico digestion.

table 2

Mr (expt) Trypsin ZT-4 Trypsin ZT-3 Trypsin ZT-2 Trypsin ZT-1 List of sequences Changes 781.5661 842.4931 1144.5651 1144.5415 1144.5415 (R)IIGGQDCEPR(S) Carbamidomethyl (C) 1173.6305 1191.6567 1191.6368 1191.6368 1191.6368 1191.6368 (R)VFEGTEQLVK(T) Guanidinyl (K) 1210.5861 1212.5237 1216.5105 1216.4908 1216.4908 (R)MVCAGYMDGGR(D) Carbamidomethyl (C) 1222.6291 1260.5201 1260.4992 1260.4992 (R)MMCVGFMDGGR(D) Carbamidomethyl (C) 1270.6331 1276.5112 1276.4941 1276.4941 (R)MMCVGFMDGGR(D) Carbamidomethyl (C)
Oxidation (M) 1638.8623 1993.9786 2635.2760 2635.2127 2635.2127 (R)DVCNGDSGSPLVC
EGVLTGLVSWGR(G) Carbamidomethyl (C)
Carboxymethyl (C) 2649.2755 2649.2283 2649.2283 (R)DVCNGDSGSPLVC
EGVLTGLVSWGR(G) Carboxymethyl (C)
Propionamid (C) 2752.4779 2762.6478 2766.5037 2776.6052 2776.3354 2776.3354 (R)LQCLDVPIVETVDC
CEAAYPGMISPR (M) 2800.4952 2800.3831 2800.3831 (R)LQCLDVPIVEPVA
CQASYPGMISPR(M) 2 Carbamidomethyl (C) 2814.5098 2814.3987 2814.3987 (R)LQCLDVPIVEPVA
CQASYPGMISPR(M) Carbamidomethyl (C)
Propionamid (C) 2830.5112 2830.3936 2830.3936 (R)LQCLDVPIVEPV
ACQASYPGMISPR(M) Carboxymethyl
Oxidative propionamide 2833.4913 2833.3569 2833.3569 (R)LQCLDVPIVETV
DCEAAYPGMISPR(M) 2 Carbamidomethyl 2847.5160 2847.3725 2847.3725 (R)LQCLDVPIVETV
DCEAAYPGMISPR(M) Carbamidomethyl (C)
Propionamid

The sequences in Table 2 are presented below:

Seq ID No Amino acid sequence SEQ ID NO 6 IIGGQDCEPR SEQ ID NO 7 VFEGTEQLVK SEQ ID NO 9 LQCLDVPIVETVDCEAAYPGMISPR SEQ ID NO 8 LQCLDVPIVEPVACQASYPGMISPR SEQ ID NO 8 LQCLDVPIVEPVACQASYPGMISPR SEQ ID NO 8 LQCLDVPIVEPVACQASYPGMISPR SEQ ID NO 9 LQCLDVPIVETVDCEAAYPGMISPR SEQ ID NO 9 LQCLDVPIVETVDCEAAYPGMISPR SEQ ID NO 10 MVCAGYMDGGR SEQ ID NO 11 MMCVGFMDGGR SEQ ID NO 11 MMCVGFMDGGR SEQ ID NO 13 DVCNGDSGSPLVCEGVLTGLVSWGR SEQ ID NO 13 DVCNGDSGSPLVCEGVLTGLVSWGR

Example 3

Multiple substrate profiling for ZT trypsin isoforms

Multiple substrate profiling has been used to analyze the substrate specificity of enzymes (proteinases) (O'Donoghue, Eroy-Reveles et al., 2012, Nat Methods 9: 1095-1100). Proteinases can cleave substrates at different amino acid residues in the peptide sequence, demonstrating differences in substrate specificity. Trypsins are identified by their preference for cleaving the C-terminus into arginine or lysine residues.

Enzyme isoforms

Cod trypsin isoenzymes were separated by applying benzamidine purified cod trypsin to a MonoQ HR 5/5 ion exchange column. To isolate trypsin I, the column was equilibrated with 20 mM Tris, 5 mM ethanolamine, 10 mM CaCl ₂ , pH 9.1, and the enzymes were eluted with a linear gradient of 0-200 mM NaCl in 80 column volumes, a linear gradient of 200-1000 mM NaCl in 10 column volumes at a flow rate of 1 ml/min. The pH of the sample was adjusted to pH 9.1 with 1 M NaOH before use.

To isolate ZT trypsin isoforms, the column was equilibrated with 25 mM Tris, 25 mM CaCl ₂ , 15% (v/v) glycerol, pH 7.5, and the enzymes were eluted with a linear gradient of 0-150 mM NaCl in 5 column volumes, linear gradient 150 -550 mM NaCl in 20 column volumes, linear gradient of 500-1000 mM NaCl in 1 column volume at a flow rate of 1 ml/min.

The composition of both enzyme samples (trypsin ZT and trypsin I) was adjusted so that the final concentration of Tris was 60 mM, CaCl ₂ 30 mM and 15% glycerol (v/v) with a pH of 8.5.

Multiple Substrate Profiling Analysis

Purified isoforms of trypsin ZT or trypsin I were incubated (5, 15 and 60 min) with a library of peptides (124 defined peptides, 14-mer sequences, total 1612 peptide bonds) with extensive physicochemical diversity, as described in O'Donoghue, Eroy- Reveles et al. (O'Donoghue, Eroy-Reveles et al., 2012, Nat Methods 9: 1095-1100). After incubation, cleavage sites were identified using mass spectrometric analysis (O'Donoghue, Eroy-Reveles et al., 2012, Nat Methods 9: 1095-1100).

results

Multiple substrate profiling analysis unexpectedly revealed that there are multiple cleavage sites unique to the ZT trypsin isoforms compared to trypsin I described in WO 2000078332 (Figure 4 and Table 4). In addition, cleavage sites were identified that showed preferential cleavage by ZT trypsin isomers compared to trypsin I (Table 4). Based on the results of the multiple substrate profiling analysis, a table was created containing numerical values based on the standard score value (Z-score) at positions P4-P4' in the substrate, where the substrate specific trypsin ZT was compared with trypsin I (Table 3). Positive values (Z-score) reflect the preference of an amino acid residue at a particular position P in favor of ZT trypsin isoforms over trypsin I. Surprisingly, multiple substrate profiling of ZT trypsin isoforms revealed that the amino acids surrounding arginine or lysine in the substrate have different effects on its cleavage compared to trypsin I.

Table 3 shows numerical values based on standard scores (Z-score) at positions P4-P4' in the substrate, compared with the substrate specificity of trypsin ZT and trypsin I after incubation for 5, 15 and 60 min. The amino acid residues in the substrate undergoing cleavage were designated P1, P2, P3, P4 in the N-terminal direction of the cleavage bond (Schechter and Berger, 1967, Biochem Biophys Res Commun 27: 157-162, Schechter and Berger, 1968, Biochem Biophys Res Commun 32: 898-902). Residues in the C-terminal direction are designated P1', P2', P3', P4'. P positions are presented in columns, and rows show amino acids at different P positions. Positive values (Z-score) in the table reflect the preference of an amino acid residue at a particular P position in favor of ZT trypsin isoforms over trypsin I. Negative values (Z-score ) reflect a preference for trypsin I over trypsin ZT. For example, based on the values in Table 3, trypsin ZT is much better adapted to cleave peptides containing multiple consecutive positively charged residues (arginine or lysine) compared to trypsin I. Again, these types of sequences, i.e., lysine and arginine rich amino acid sequences are often found in protein toxins, proteins and peptides involved in pathogenesis (Suzuki, Orba et al., 2010, PLoS Pathog 6: e1000801, Fentress, Steinfeldt et al., 2012, Cell Microbiol 14: 1921-1933, Jiang , Cun et al., 2012, J Virol 86: 7256-7267, Milletti, 2012, Drug Discov Today 17: 850-860, Gallaher and Garry, 2015, Viruses 7: 285-305). As mentioned earlier, ZT trypsin isoforms have properties that are advantageous to proteins responsible for the pathogenicity of many microorganisms.

Table 3.

5 minutes. Amino acid Position P4 P3 P2 P1 P1´ P2´ P3´ P4´ G -0.60 0.88 0.48 0.26 -1.21 0.11 0.97 0.08 A 0.01 0.48 1.92 -0.57 2.31 1.01 -0.62 -0.14 S -0.06 -0.04 0.43 0.23 0.25 -0.52 -1.47 0.32 P -1.04 -0.59 0.39 -0.16 0.25 1.86 -0.30 -0.50 V 0.99 -1.14 -1.88 0.23 0.43 0.54 0.01 0.38 T 0.05 -1.40 0.43 0.72 0.48 -0.02 -1.39 -0.06 L -0.50 0.48 -2.26 0.25 -0.75 -0.43 0.88 0.91 I 1.42 -0.51 -0.31 0.72 -0.67 -0.36 -0.04 -0.62 N 0.95 0.93 -0.47 -0.15 -1.33 -0.79 -0.56 0.02 D 0.33 0.43 0.24 -0.48 -0.02 -0.53 -0.52 -0.69 Q -1.38 -0.89 -0.87 0.70 -0.01 -0.55 -0.01 1.10 K -0.12 0.38 1.88 -6.09 1.02 -1.11 -0.14 -0.27 E 0.38 -0.66 -0.96 0.30 -0.53 -1.06 0.48 -0.09 M -1.91 -1.27 -0.28 0.25 -0.79 -0.86 0.33 0.38 H -0.09 0.38 0.92 0.25 -0.39 0.58 0.38 1.49 F 0.49 -0.41 -0.26 0.72 -0.01 -0.42 -0.58 0.10 R -0.69 -0.09 -0.07 2.69 1.95 0.99 0.12 0.27 Y -0.60 0.75 1.04 0.23 -0.15 -0.42 0.95 -0.12 W 1.00 0.64 -0.84 -0.15 -0.59 1.00 1.36 -0.53

Table 3 continued.

15 minutes. Amino acid Position P4 P3 P2 P1 P1´ P2´ P3´ P4´ G 0.33 1.65 -0.27 0.48 -0.64 0.60 1.79 -0.36 A -0.34 -0.66 1.33 -0.52 1.88 -0.60 -1.05 0.17 S 0.30 -0.41 0.73 0.44 0.51 0.03 -1.16 0.55 P 0.26 -0.62 0.23 0.12 0.54 1.70 0.66 -0.79 V 0.55 -0.08 -0.75 0.44 0.36 0.97 0.97 0.81 T 0.04 -0.25 -0.50 0.89 0.44 0.81 -1.21 -0.63 L 0.49 0.11 -1.57 0.18 0.18 0.44 0.73 0.73 I -1.07 -0.89 -0.97 0.44 -0.54 0.12 0.32 0.04 N -0.86 0.81 0.96 0.89 -1.50 0.67 -1.74 -0.89 D -1.45 -0.43 0.07 -0.03 0.11 -2.02 0.42 -0.76 Q -0.32 -0.71 -1.07 0.89 -0.28 -0.49 0.43 0.73 K -0.72 -0.16 1.35 -6.74 0.96 -1.11 0.25 0.48 E 0.25 -0.72 -0.01 0.54 -0.39 -1.35 -1.49 0.25 M -0.89 -0.59 -0.16 0.47 0.25 -2.23 0.19 1.55 H 0.33 0.26 1.26 0.89 -0.67 0.29 -0.19 0.40 F 1.34 0.15 -0.82 0.89 -0.33 -0.36 -0.26 0.20 R -0.67 -0.17 -0.82 -1.11 2.17 1.35 0.65 -0.78 Y 0.30 0.40 0.58 0.44 -1.00 0.57 0.89 -1.07 W 0.49 -0.26 -1.38 0.13 -1.83 -0.57 0.36 0.50

Table 3 continued.

60 min. Amino acid Position P4 P3 P2 P1 P1´ P2´ P3´ P4´ G 0.45 0.80 -1.05 0.26 -0.27 0.71 0.65 -0.93 A 0.05 -0.74 1.65 -0.30 2.33 0.39 -0.43 -1.56 S -0.30 0.57 0.52 0.52 0.60 -1.19 -0.08 0.59 P -0.72 0.42 -1.24 -0.05 0.90 2.28 -0.28 -0.57 V -0.25 0.51 -0.09 0.84 -0.55 1.03 -0.19 0.91 T 0.49 -0.07 0.91 0.52 -0.54 0.80 -1.21 0.13 L -0.23 -0.47 -0.73 -0.06 0.66 0.22 0.41 0.38 I -0.73 -0.53 0.04 0.20 0.44 0.20 0.85 0.61 N -0.90 0.15 0.28 -0.05 -1.49 -0.43 -1.18 -0.18 D -1.33 -0.03 -0.16 1.32 -0.70 -1.42 -0.26 0.67 Q 0.63 -0.44 -1.36 0.85 0.33 -0.17 -1.15 1.09 K 0.43 -0.02 1.24 -7.02 0.97 -1.46 0.73 0.90 E -0.01 -1.45 -0.21 0.54 -1.36 -1.64 -1.11 -0.36 M -1.39 -0.36 0.01 0.24 -0.24 -0.93 -0.37 0.79 H 0.79 -1.38 1.23 0.85 -1.06 -0.30 0.47 0.57 F 0.48 0.15 -1.67 0.52 -0.06 0.19 0.85 -0.07 R -0.46 -0.02 -0.91 -0.91 1.67 0.91 1.50 -0.11 Y 0.73 0.41 0.25 0.52 -1.36 0.58 1.60 -2.25 W 0.85 1.02 0.09 0.90 -0.21 0.03 -0.49 -0.42

Table 4 shows the precursor ion intensities of selected peptides from multiple profiling after incubation with trypsin ZT or trypsin I. Precursor ion intensities represent the intensities of the peptide peaks in the MS before fragmentation, which represent a measure of the relative abundance for a given species. X in the peptide sequence denotes a placeholder for the absence of an amino acid in the peptide sequence. Data show that ZT trypsin isoforms are able to exclusively cleave three peptide sequences (QGKKAPXX, WGNRSPLE and QAVRPNGM) compared to trypsin I. Additionally, data show that ZT trypsin isoforms preferentially cleave two peptides compared to trypsin I. ZT trypsin isoforms for cleavage of XIARQPWN within 5 min and FDNRVGKW within 15 min, whereas cleavage within these two peptides was detected only with trypsin I after 60 min incubation.

Table 4

List of sequences SEQ ID NO: Precursor Ion Intensity Trypsin ZT Trypsin I 5' 15' 60' 5' 15' 60' QGKKAPXX SEQ ID NO 14 175343 278749 256859 0 0 0 WGNRSPLE SEQ ID NO 15 21152 42866 47692 0 0 0 QAVRPNGM SEQ ID NO 16 0 12772 29668 0 0 0 FDNRVGKW SEQ ID NO 17 0 187219 937228 0 0 164917 XIARQPWN SEQ ID NO 18 130793 0 350390 0 0 188625

Example 4

Purification of cod ZT trypsin isoforms from concentrated cod viscera extract

About 12 liters of the ultrafiltered and diafiltered concentrate obtained in example 1 were applied to a continuous connected series of about 1 liter chromatography columns, the first of which contained a fast soluble cation exchange resin CM (GE healthcare), the second - the affinity ligand p-aminobenzamidine bound to Sepharose resin (GE healthcare), and the third DEAE instant anion exchange resin (GE healthcare). CM columns and p-aminobenzamidine columns were pre-equilibrated with about 10 column volumes of 25 mM Tris buffer, pH 7.8, containing 2.5 mM calcium chloride. The concentrate was pumped into SM columns and p-aminobenzamidine columns at a flow rate of about 100 ml/min. When the application of the concentrated solution to the columns was completed, the residual material was washed off the continuous column system with about 8 liters of Buffer A. Once the wash was complete, the p-aminobenzamidine affinity column was disconnected from the CM column and washed with about 5 column volumes of 25 mM Tris high saline. -buffer with pH 7.5 containing 0.5 M NaCl and 2.5 mM calcium chloride. The cod trypsins were then desorbed from the affinity ligand and the column was eluted with an acidic solution of 25 mM acetic acid, pH 3.2, containing 10 mM calcium chloride and 30% glycerol. The cod trypsin fraction was collected in neutralization buffer 200 mM Tris, pH 8.5, containing 30% glycerol. The DEAE anion exchange column was pre-equilibrated with about 10 column volumes of 25 mM Tris buffer, pH 7.8, containing 2.5 mM calcium chloride. The cod trypsin fraction was run on a DEAE anion exchange resin and washed with approximately 5 column volumes of equilibration buffer. ZT trypsin isoforms were then desorbed from the column using a 0.95 M NaCl gradient salt solution. The purified preparation of cod trypsin ZT isoform was homogenized by SDS PAGE and FPLC Mono Q chromatography. The purified preparation is sterilized by filtration through a 0.22-micron filter and stored frozen at a temperature of about -20°C.

Example 5

Preparation of a hydrogel preparation of cod trypsin, including cod ZT trypsin isoforms

Purified cod ZT trypsin isoform containing the preparation of Example 2 was mixed with a hydrocolloid gel containing an aqueous gel containing 0.8% w/v. Carbomer 940 and 40% glycerol. The cod trypsin preparation was mixed in a 1:1 ratio with the hydrogel to obtain a final concentration of 4 enzyme units per mg (U/mg) of the final gel-enzyme mixture (enzyme hydrogel ointment), the enzyme unit was determined using Cbz-GPR-pNA as a substrate as described above. Thus, the resulting enzyme hydrogel ointment contained approximately 0.01 mg/ml or 1 U/ml cod trypsin enzymes, 0.4% carbomer 940, 20% glycerol and 0.04% paraoxybenzoate.

Example 6

Antiviral activity of ZT trypsin isoforms against enterovirus as a model rhinovirus

Picornaviruses are small, single-stranded positive sense RNA viruses that lack a lipid envelope. Enterovirus and rhinovirus are two genera of Picornaviridae. Enterovirus replicates initially in the oropharynx and survives in the acidic environment of the stomach. The small intestine is the main site of enterovirus invasion. Rhinovirus replicates in the nasal passage but is destroyed in the acidic environment of the stomach. Rhinovirus and enterovirus have identical morphology but can be differentiated based on clinical, biophysical and epidemiological studies. Rhinovirus is problematic for circulation in cell cultures (Racaniello, 2007, Fields virology, 5th ed: 795-838). Therefore, enterovirus was used as a rhinovirus model to analyze the antiviral properties of ZT trypsin isoforms.

Materials and methods

Enterovirus stock solution

Enterovirus (Coxsackievirus B2) was isolated from a patient at the University Hospital of Iceland, Department of Virology and grown in MA-104 cells (African green monkey kidney cells) or Vero cells (African green monkey kidney cells). Coxsackievirus B2 was titrated (TCID ₅₀ = 8.23) according to Reed-Muench (REED and MUENCH, 1938, American Journal of Epidemiology 27: 493-497).

Enzyme isoforms

Cod trypsin isoenzymes were separated by applying benzamidine purified cod trypsin to a MonoQ HR 5/5 ion exchange column. To isolate ZT trypsin isoforms, the column was equilibrated with 25 mM Tris, 25 mM CaCl ₂ , pH 7.5, and the enzymes were eluted with a linear 0-400 mM NaCl gradient in 5 column volumes, linear 400-1000 mM NaCl in 20 at a flow volume of 1 ml/ min.

Treatment

MA-104 cells were grown in a 96-well microtiter plate to 95% confluence. Enterovirus was diluted from the stock solution to 10 ^-6 using minimal essential medium (MEM) and 1.5 ml of the diluted solution was placed in two different tubes. The tubes were treated with 1.5 ml of the ZT isoform of trypsin and 1.5 ml of MEM as a positive control. The negative control was MEM (3 ml) without virus. All vials were incubated at 34°C and 5% CO ₂ for 90 min before adding 100 μl of benzamidine agarose solution to remove residual trypsin and incubating for a further 30 min. The solution was centrifuged at 5500 rpm for 3 min before placing 2 ml of supernatant into an Eppendorf tube. 200 μl of solution was placed in each well, for a total of 10 wells per microtiter plate. The plate was incubated for 30 min. After incubation, the medium was discarded and MEM with 1% newborn calf serum (NCS) was added to all wells. A microscope was used to monitor the presence of infection.

results

ZT trypsin isoforms were very effective as antiviral agents against enterovirus (Coxsackievirus B2), which was used as a model for rhinovirus. The results are presented in Fig. 5.

--->

List of sequences

<110> Zymetech EHF

<120> NOVEL TRYPSIN ISOFORMS AND THEIR USE

<130> 21083149

<150> EP15178208.3

<151> 2015-07-24

<160> 23

<170> Patent In version 3.5

<210> 1

<211> 227

<212>PRT

<213> Artificial Sequence

<220>

<223> Generic sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa can be I or V

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa can be Q or H

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Xaa can be D or E

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Xaa can be R or N

<220>

<221> MISC_FEATURE

<222> (77)..(77)

<223> Xaa is L

<220>

<221> MISC_FEATURE

<222> (148)..(148)

<223> Xaa can be T or P

<220>

<221> MISC_FEATURE

<222> (150)..(150)

<223> Xaa can be D or A

<220>

<221> MISC_FEATURE

<222> (152)..(152)

<223> Xaa can be E or Q

<220>

<221> MISC_FEATURE

<222> (154)..(154)

<223> Xaa can be A or S

<220>

<221> MISC_FEATURE

<222> (164)..(164)

<223> Xaa can be V or M

<220>

<221> MISC_FEATURE

<222> (166)..(166)

<223> Xaa can be A or V

<220>

<221> MISC_FEATURE

<222> (168)..(168)

<223> Xaa can be Y or F

<220>

<221> MISC_FEATURE

<222> (175)..(175)

<223> Xaa can be A or V

<220>

<221> MISC_FEATURE

<222> (198)..(198)

<223> Xaa can be Q or R

<220>

<221> MISC_FEATURE

<222> (202)..(202)

<223> Xaa can be L or E

<220>

<221> MISC_FEATURE

<222> (205)..(205)

<223> Xaa can be Y or S

<220>

<221> MISC_FEATURE

<222> (215)..(215)

<223> Xaa can be Y or F

<400> 1

Ile Xaa Gly Gly Xaa Xaa Cys Glu Pro Xaa Ser Arg Pro Phe Met Ala

1 5 10 15

Ser Leu Asn Tyr Gly Tyr His Phe Cys Gly Gly Val Leu Ile Asn Asp

20 25 30

Gln Trp Val Leu Ser Val Ala His Cys Trp Tyr Asn Pro Tyr Tyr Met

35 40 45

Gln Val Met Leu Gly Glu His Asp Leu Arg Val Phe Glu Gly Thr Glu

50 55 60

Gln Leu Val Lys Thr Asn Thr Ile Phe Trp His Glu Xaa Tyr Asp Tyr

65 70 75 80

Gln Thr Leu Asp Tyr Asp Met Met Met Ile Lys Leu Tyr His Pro Val

85 90 95

Glu Val Thr Gln Ser Val Ala Pro Ile Ser Leu Pro Thr Gly Pro Pro

100 105 110

Asp Gly Gly Met Leu Cys Ser Val Ser Gly Trp Gly Asn Met Ala Met

115 120 125

Gly Glu Glu Val Asn Leu Pro Thr Arg Leu Gln Cys Leu Asp Val Pro

130 135 140

Ile Val Glu Xaa Val Xaa Cys Xaa Ala Xaa Tyr Pro Gly Met Ile Ser

145 150 155 160

Pro Arg Met Xaa Cys Xaa Gly Xaa Met Asp Gly Gly Arg Asp Xaa Cys

165 170 175

Asn Gly Asp Ser Gly Ser Pro Leu Val Cys Glu Gly Val Leu Thr Gly

180 185 190

Leu Val Ser Trp Gly Xaa Gly Cys Ala Xaa Pro Asn Xaa Pro Gly Val

195 200 205

Tyr Val Lys Val Tyr Glu Xaa Leu Ser Trp Ile Gln Thr Thr Leu Asp

210 215 220

Ala Asn Pro

225

<210> 2

<211> 227

<212>PRT

<213> Gadus morhua

<400> 2

Ile Val Gly Gly His Glu Cys Glu Pro Asn Ser Arg Pro Phe Met Ala

1 5 10 15

Ser Leu Asn Tyr Gly Tyr His Phe Cys Gly Gly Val Leu Ile Asn Asp

20 25 30

Gln Trp Val Leu Ser Val Ala His Cys Trp Tyr Asn Pro Tyr Tyr Met

35 40 45

Gln Val Met Leu Gly Glu His Asp Leu Arg Val Phe Glu Gly Thr Glu

50 55 60

Gln Leu Val Lys Thr Asn Thr Ile Phe Trp His Glu Leu Tyr Asp Tyr

65 70 75 80

Gln Thr Leu Asp Tyr Asp Met Met Met Ile Lys Leu Tyr His Pro Val

85 90 95

Glu Val Thr Gln Ser Val Ala Pro Ile Ser Leu Pro Thr Gly Pro Pro

100 105 110

Asp Gly Gly Met Leu Cys Ser Val Ser Gly Trp Gly Asn Met Ala Met

115 120 125

Gly Glu Glu Val Asn Leu Pro Thr Arg Leu Gln Cys Leu Asp Val Pro

130 135 140

Ile Val Glu Pro Val Ala Cys Gln Ala Ser Tyr Pro Gly Met Ile Ser

145 150 155 160

Pro Arg Met Met Cys Val Gly Phe Met Asp Gly Gly Arg Asp Val Cys

165 170 175

Asn Gly Asp Ser Gly Ser Pro Leu Val Cys Glu Gly Val Leu Thr Gly

180 185 190

Leu Val Ser Trp Gly Arg Gly Cys Ala Glu Pro Asn Ser Pro Gly Val

195 200 205

Tyr Val Lys Val Tyr Glu Phe Leu Ser Trp Ile Gln Thr Thr Leu Asp

210 215 220

Ala Asn Pro

225

<210> 3

<211> 227

<212>PRT

<213> Gadus morhua

<400> 3

Ile Val Gly Gly His Glu Cys Glu Pro Asn Ser Arg Pro Phe Met Ala

1 5 10 15

Ser Leu Asn Tyr Gly Tyr His Phe Cys Gly Gly Val Leu Ile Asn Asp

20 25 30

Gln Trp Val Leu Ser Val Ala His Cys Trp Tyr Asn Pro Tyr Tyr Met

35 40 45

Gln Val Met Leu Gly Glu His Asp Leu Arg Val Phe Glu Gly Thr Glu

50 55 60

Gln Leu Val Lys Thr Asn Thr Ile Phe Trp His Glu Leu Tyr Asp Tyr

65 70 75 80

Gln Thr Leu Asp Tyr Asp Met Met Met Ile Lys Leu Tyr His Pro Val

85 90 95

Glu Val Thr Gln Ser Val Ala Pro Ile Ser Leu Pro Thr Gly Pro Pro

100 105 110

Asp Gly Gly Met Leu Cys Ser Val Ser Gly Trp Gly Asn Met Ala Met

115 120 125

Gly Glu Glu Val Asn Leu Pro Thr Arg Leu Gln Cys Leu Asp Val Pro

130 135 140

Ile Val Glu Thr Val Asp Cys Glu Ala Ala Tyr Pro Gly Met Ile Ser

145 150 155 160

Pro Arg Met Val Cys Ala Gly Tyr Met Asp Gly Gly Arg Asp Ala Cys

165 170 175

Asn Gly Asp Ser Gly Ser Pro Leu Val Cys Glu Gly Val Leu Thr Gly

180 185 190

Leu Val Ser Trp Gly Gln Gly Cys Ala Leu Pro Asn Tyr Pro Gly Val

195 200 205

Tyr Val Lys Val Tyr Glu Tyr Leu Ser Trp Ile Gln Thr Thr Leu Asp

210 215 220

Ala Asn Pro

225

<210> 4

<211> 227

<212>PRT

<213> Gadus morhua

<400> 4

Ile Ile Gly Gly Gln Asp Cys Glu Pro Arg Ser Arg Pro Phe Met Ala

1 5 10 15

Ser Leu Asn Tyr Gly Tyr His Phe Cys Gly Gly Val Leu Ile Asn Asp

20 25 30

Gln Trp Val Leu Ser Val Ala His Cys Trp Tyr Asn Pro Tyr Tyr Met

35 40 45

Gln Val Met Leu Gly Glu His Asp Leu Arg Val Phe Glu Gly Thr Glu

50 55 60

Gln Leu Val Lys Thr Asn Thr Ile Phe Trp His Glu Leu Tyr Asp Tyr

65 70 75 80

Gln Thr Leu Asp Tyr Asp Met Met Met Ile Lys Leu Tyr His Pro Val

85 90 95

Glu Val Thr Gln Ser Val Ala Pro Ile Ser Leu Pro Thr Gly Pro Pro

100 105 110

Asp Gly Gly Met Leu Cys Ser Val Ser Gly Trp Gly Asn Met Ala Met

115 120 125

Gly Glu Glu Val Asn Leu Pro Thr Arg Leu Gln Cys Leu Asp Val Pro

130 135 140

Ile Val Glu Pro Val Ala Cys Gln Ala Ser Tyr Pro Gly Met Ile Ser

145 150 155 160

Pro Arg Met Met Cys Val Gly Phe Met Asp Gly Gly Arg Asp Val Cys

165 170 175

Asn Gly Asp Ser Gly Ser Pro Leu Val Cys Glu Gly Val Leu Thr Gly

180 185 190

Leu Val Ser Trp Gly Arg Gly Cys Ala Glu Pro Asn Ser Pro Gly Val

195 200 205

Tyr Val Lys Val Tyr Glu Phe Leu Ser Trp Ile Gln Thr Thr Leu Asp

210 215 220

Ala Asn Pro

225

<210> 5

<211> 227

<212>PRT

<213> Gadus morhua

<400> 5

Ile Ile Gly Gly Gln Asp Cys Glu Pro Arg Ser Arg Pro Phe Met Ala

1 5 10 15

Ser Leu Asn Tyr Gly Tyr His Phe Cys Gly Gly Val Leu Ile Asn Asp

20 25 30

Gln Trp Val Leu Ser Val Ala His Cys Trp Tyr Asn Pro Tyr Tyr Met

35 40 45

Gln Val Met Leu Gly Glu His Asp Leu Arg Val Phe Glu Gly Thr Glu

50 55 60

Gln Leu Val Lys Thr Asn Thr Ile Phe Trp His Glu Leu Tyr Asp Tyr

65 70 75 80

Gln Thr Leu Asp Tyr Asp Met Met Met Ile Lys Leu Tyr His Pro Val

85 90 95

Glu Val Thr Gln Ser Val Ala Pro Ile Ser Leu Pro Thr Gly Pro Pro

100 105 110

Asp Gly Gly Met Leu Cys Ser Val Ser Gly Trp Gly Asn Met Ala Met

115 120 125

Gly Glu Glu Val Asn Leu Pro Thr Arg Leu Gln Cys Leu Asp Val Pro

130 135 140

Ile Val Glu Thr Val Asp Cys Glu Ala Ala Tyr Pro Gly Met Ile Ser

145 150 155 160

Pro Arg Met Val Cys Ala Gly Tyr Met Asp Gly Gly Arg Asp Ala Cys

165 170 175

Asn Gly Asp Ser Gly Ser Pro Leu Val Cys Glu Gly Val Leu Thr Gly

180 185 190

Leu Val Ser Trp Gly Gln Gly Cys Ala Leu Pro Asn Tyr Pro Gly Val

195 200 205

Tyr Val Lys Val Tyr Glu Tyr Leu Ser Trp Ile Gln Thr Thr Leu Asp

210 215 220

Ala Asn Pro

225

<210> 6

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 1

<400> 6

Ile Ile Gly Gly Gln Asp Cys Glu Pro Arg

1 5 10

<210> 7

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 2

<400> 7

Val Phe Glu Gly Thr Glu Gln Leu Val Lys

1 5 10

<210> 8

<211> 25

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 3

<400> 8

Leu Gln Cys Leu Asp Val Pro Ile Val Glu Pro Val Ala Cys Gln Ala

1 5 10 15

Ser Tyr Pro Gly Met Ile Ser Pro Arg

20 25

<210> 9

<211> 25

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 4

<400> 9

Leu Gln Cys Leu Asp Val Pro Ile Val Glu Thr Val Asp Cys Glu Ala

1 5 10 15

Ala Tyr Pro Gly Met Ile Ser Pro Arg

20 25

<210> 10

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 5

<400> 10

Met Val Cys Ala Gly Tyr Met Asp Gly Gly Arg

1 5 10

<210> 11

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 6

<400> 11

Met Met Cys Val Gly Phe Met Asp Gly Gly Arg

1 5 10

<210> 12

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 7

<400> 12

Gly Cys Ala Glu Pro Asn Ser Pro Gly Val Tyr Val Lys

1 5 10

<210> 13

<211> 25

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 8

<400> 13

Asp Val Cys Asn Gly Asp Ser Gly Ser Pro Leu Val Cys Glu Gly Val

1 5 10 15

Leu Thr Gly Leu Val Ser Trp Gly Arg

20 25

<210> 14

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 9

<400> 14

Gln Gly Lys Lys Ala Pro

15

<210> 15

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 10

<400> 15

Trp Gly Asn Arg Ser Pro Leu Glu

15

<210> 16

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 11

<400> 16

Gln Ala Val Arg Pro Asn Gly Met

15

<210> 17

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 12

<400> 17

Phe Asp Asn Arg Val Gly Lys Trp

15

<210> 18

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> Fragment 13

<400> 18

Ile Ala Arg Gln Pro Trp Asn

15

<210> 19

<211> 250

<212>PRT

<213> Gadus morhua

<400> 19

Met Ile Gly Leu Ala Leu Leu Met Leu Leu Gly Ala Ala Ala Ala Ala

1 5 10 15

Val Pro Arg Asp Val Gly Lys Ile Val Gly Gly His Glu Cys Glu Pro

20 25 30

Asn Ser Arg Pro Phe Met Ala Ser Leu Asn Tyr Gly Tyr His Phe Cys

35 40 45

Gly Gly Val Leu Ile Asn Asp Gln Trp Val Leu Ser Val Ala His Cys

50 55 60

Trp Tyr Asn Pro Tyr Tyr Met Gln Val Met Leu Gly Glu His Asp Leu

65 70 75 80

Arg Val Phe Glu Gly Thr Glu Gln Leu Val Lys Thr Asn Thr Ile Phe

85 90 95

Trp His Glu Leu Tyr Asp Tyr Gln Thr Leu Asp Tyr Asp Met Met Met

100 105 110

Ile Lys Leu Tyr His Pro Val Glu Val Thr Gln Ser Val Ala Pro Ile

115 120 125

Ser Leu Pro Thr Gly Pro Pro Asp Gly Gly Met Leu Cys Ser Val Ser

130 135 140

Gly Trp Gly Asn Met Ala Met Gly Glu Glu Val Asn Leu Pro Thr Arg

145 150 155 160

Leu Gln Cys Leu Asp Val Pro Ile Val Glu Pro Val Ala Cys Gln Ala

165 170 175

Ser Tyr Pro Gly Met Ile Ser Pro Arg Met Met Cys Val Gly Phe Met

180 185 190

Asp Gly Gly Arg Asp Val Cys Asn Gly Asp Ser Gly Ser Pro Leu Val

195 200 205

Cys Glu Gly Val Leu Thr Gly Leu Val Ser Trp Gly Arg Gly Cys Ala

210 215 220

Glu Pro Asn Ser Pro Gly Val Tyr Val Lys Val Tyr Glu Phe Leu Ser

225 230 235 240

Trp Ile Gln Thr Thr Leu Asp Ala Asn Pro

245 250

<210> 20

<211> 250

<212>PRT

<213> Gadus morhua

<400> 20

Met Ile Gly Leu Ala Leu Leu Met Leu Leu Gly Ala Ala Ala Ala Ala

1 5 10 15

Val Pro Arg Asp Val Gly Lys Ile Val Gly Gly His Glu Cys Glu Pro

20 25 30

Asn Ser Arg Pro Phe Met Ala Ser Leu Asn Tyr Gly Tyr His Phe Cys

35 40 45

Gly Gly Val Leu Ile Asn Asp Gln Trp Val Leu Ser Val Ala His Cys

50 55 60

Trp Tyr Asn Pro Tyr Tyr Met Gln Val Met Leu Gly Glu His Asp Leu

65 70 75 80

Arg Val Phe Glu Gly Thr Glu Gln Leu Val Lys Thr Asn Thr Ile Phe

85 90 95

Trp His Glu Leu Tyr Asp Tyr Gln Thr Leu Asp Tyr Asp Met Met Met

100 105 110

Ile Lys Leu Tyr His Pro Val Glu Val Thr Gln Ser Val Ala Pro Ile

115 120 125

Ser Leu Pro Thr Gly Pro Pro Asp Gly Gly Met Leu Cys Ser Val Ser

130 135 140

Gly Trp Gly Asn Met Ala Met Gly Glu Glu Val Asn Leu Pro Thr Arg

145 150 155 160

Leu Gln Cys Leu Asp Val Pro Ile Val Glu Thr Val Asp Cys Glu Ala

165 170 175

Ala Tyr Pro Gly Met Ile Ser Pro Arg Met Val Cys Ala Gly Tyr Met

180 185 190

Asp Gly Gly Arg Asp Ala Cys Asn Gly Asp Ser Gly Ser Pro Leu Val

195 200 205

Cys Glu Gly Val Leu Thr Gly Leu Val Ser Trp Gly Gln Gly Cys Ala

210 215 220

Leu Pro Asn Tyr Pro Gly Val Tyr Val Lys Val Tyr Glu Tyr Leu Ser

225 230 235 240

Trp Ile Gln Thr Thr Leu Asp Ala Asn Pro

245 250

<210> 21

<211> 249

<212>PRT

<213> Gadus morhua

<400> 21

Met Ile Gly Leu Ala Leu Leu Met Leu Leu Gly Ala Ala Ala Ala Val

1 5 10 15

Pro Arg Glu Asp Gly Arg Ile Ile Gly Gly Gln Asp Cys Glu Pro Arg

20 25 30

Ser Arg Pro Phe Met Ala Ser Leu Asn Tyr Gly Tyr His Phe Cys Gly

35 40 45

Gly Val Leu Ile Asn Asp Gln Trp Val Leu Ser Val Ala His Cys Trp

50 55 60

Tyr Asn Pro Tyr Tyr Met Gln Val Met Leu Gly Glu His Asp Leu Arg

65 70 75 80

Val Phe Glu Gly Thr Glu Gln Leu Val Lys Thr Asn Thr Ile Phe Trp

85 90 95

His Glu Leu Tyr Asp Tyr Gln Thr Leu Asp Tyr Asp Met Met Met Ile

100 105 110

Lys Leu Tyr His Pro Val Glu Val Thr Gln Ser Val Ala Pro Ile Ser

115 120 125

Leu Pro Thr Gly Pro Pro Asp Gly Gly Met Leu Cys Ser Val Ser Gly

130 135 140

Trp Gly Asn Met Ala Met Gly Glu Glu Val Asn Leu Pro Thr Arg Leu

145 150 155 160

Gln Cys Leu Asp Val Pro Ile Val Glu Pro Val Ala Cys Gln Ala Ser

165 170 175

Tyr Pro Gly Met Ile Ser Pro Arg Met Met Cys Val Gly Phe Met Asp

180 185 190

Gly Gly Arg Asp Val Cys Asn Gly Asp Ser Gly Ser Pro Leu Val Cys

195 200 205

Glu Gly Val Leu Thr Gly Leu Val Ser Trp Gly Arg Gly Cys Ala Glu

210 215 220

Pro Asn Ser Pro Gly Val Tyr Val Lys Val Tyr Glu Phe Leu Ser Trp

225 230 235 240

Ile Gln Thr Thr Leu Asp Ala Asn Pro

245

<210> 22

<211> 249

<212>PRT

<213> Gadus morhua

<400> 22

Met Ile Gly Leu Ala Leu Leu Met Leu Leu Gly Ala Ala Ala Ala Val

1 5 10 15

Pro Arg Glu Asp Gly Arg Ile Ile Gly Gly Gln Asp Cys Glu Pro Arg

20 25 30

Ser Arg Pro Phe Met Ala Ser Leu Asn Tyr Gly Tyr His Phe Cys Gly

35 40 45

Gly Val Leu Ile Asn Asp Gln Trp Val Leu Ser Val Ala His Cys Trp

50 55 60

Tyr Asn Pro Tyr Tyr Met Gln Val Met Leu Gly Glu His Asp Leu Arg

65 70 75 80

Val Phe Glu Gly Thr Glu Gln Leu Val Lys Thr Asn Thr Ile Phe Trp

85 90 95

His Glu Leu Tyr Asp Tyr Gln Thr Leu Asp Tyr Asp Met Met Met Ile

100 105 110

Lys Leu Tyr His Pro Val Glu Val Thr Gln Ser Val Ala Pro Ile Ser

115 120 125

Leu Pro Thr Gly Pro Pro Asp Gly Gly Met Leu Cys Ser Val Ser Gly

130 135 140

Trp Gly Asn Met Ala Met Gly Glu Glu Val Asn Leu Pro Thr Arg Leu

145 150 155 160

Gln Cys Leu Asp Val Pro Ile Val Glu Thr Val Asp Cys Glu Ala Ala

165 170 175

Tyr Pro Gly Met Ile Ser Pro Arg Met Val Cys Ala Gly Tyr Met Asp

180 185 190

Gly Gly Arg Asp Ala Cys Asn Gly Asp Ser Gly Ser Pro Leu Val Cys

195 200 205

Glu Gly Val Leu Thr Gly Leu Val Ser Trp Gly Gln Gly Cys Ala Leu

210 215 220

Pro Asn Tyr Pro Gly Val Tyr Val Lys Val Tyr Glu Tyr Leu Ser Trp

225 230 235 240

Ile Gln Thr Thr Leu Asp Ala Asn Pro

245

<210> 23

<211> 12

<212>PRT

<213> Gadus morhua

<400> 23

Cys Val Leu Ser Gly Trp Val Arg Asp Thr Met Ala

1 5 10

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Claims (11)

1. An isolated isoform of Atlantic cod trypsin ZT, containing an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence with 99% or more sequence homology. 2. A medical composition having antiviral activity against picornaviruses, containing at least one isoform of Atlantic cod trypsin ZT according to claim 1, together with suitable pharmaceutical excipients and carriers. 3. The composition according to claim 2, in which said isoform(s) of Atlantic cod ZT trypsin is in a mixture with other Atlantic cod trypsins. 4. The composition of claim 2 or 3, wherein said Atlantic cod ZT trypsin isoform(s) constitute at least 5% (w/w) of the total Atlantic cod trypsin content of said composition. 5. Composition according to any one of paragraphs. 2-4, which further contains a polyvalent alcohol such as glycerin. 6. Composition according to any one of paragraphs. 2-5, which is administered topically as a lotion, hydrogel, oral spray or nasal spray. 7. Composition according to any one of paragraphs. 2-6, wherein picornavirus is a rhinovirus. 8. Use of the composition according to any one of paragraphs. 2-6 for the treatment or prevention of upper respiratory tract diseases caused by picornaviruses. 9. Use according to claim 8, wherein the picornavirus is a rhinovirus. 10. A method for treating diseases caused by picornaviruses, which includes administering a therapeutically effective amount of the composition according to any one of claims. 2-6 to the patient in need. 11. The method according to claim 10, where the picornavirus is a rhinovirus.